Skeletal muscle provides unique opportunities for investigations of myogenesis in the embryo and in regenerating muscle. Regulatory genes and signals controlling the specification and differentiation of skeletal myogenic progenitors are now well known, and cell and embryo models for investigations of these stages of myogenesis are available. Less well understood are the genetic and epigenetic mechanisms that control the earlier processes of skeletal myogenesis, including mesoderm specification and commitment of myogenic progenitors to the skeletal muscle lineage. This project utilizes unique embryonic stem cell and mesodermal cell models together with gene expression and embryo and muscle engraftment technologies to identify and characterize genes, signals, and epigenetic modifications that control early myogenesis in the mouse. Proposed studies include investigations of Zic1 and Zic2, newly discovered myogenic regulatory genes that control Myf5 activation for specification of epaxial muscle progenitors in somatic mesoderm. Upstream mesoderm regulators of Zic1 and Zic2 will be identified in gene expression and signaling studies using mesoderm explant assays. Chromatin immunoprecipitation (ChIP) and mass spectrometry proteomics tools will be used to characterize Zic, Gli, and Tcf transcription complexes and their interactions with the Myf5 epaxial somite (ES) transcriptional enhancer, which controls Myf5 activation in response to Hedgehog and Wnt signaling. A mouse embryonic stem cell (mESC) model will be used in combination with expression, shRNA knockdown, array profiling, cell culture, and chick embryo and muscle engraftment approaches to identify genes and signals that control the specifications of mesoderm and committed myogenic cell precursors. Epigenetic mechanisms that control lineage commitment will be investigated using induced pluripotent stem cell (iPSC) reprogramming together with cell culture and chick embryo and muscle engraftment. Investigations focus on epigenetic processes, including MyoD autoregulation, MyoD core enhancer DNA methylation, MyoD histone H3.3 binding, and mesoderm and myogenic lineage commitment, in myogenic cells responding to iPSC reprogramming. Effects of DNA methyltransferase and HDAC inhibitors on muscle cell reprogramming also will reveal underlying molecular mechanisms that establish and maintain the epigenetic control systems that are stable to iPSC reprogramming. These studies will contribute new knowledge of the genetic and epigenetic mechanisms that program the genomes of pluri- and multipotent embryonic cells to generate lineage- restricted stem cells for tissue differentiation and regeneration, and to the development of stem cell technologies for the treatment of muscle damage, disease, and aging. PUBLIC HEALTH RELEVANCE: Realizing the potential of stem cell biology for regenerative medicine requires a deeper understanding of the fundamental molecular and epigenetic mechanisms that program the genomes of pluri- and multipotential embryonic cells to generate committed stem cell lineages that form the tissues and organ systems of the adult. Skeletal muscle stem cell biology provides unique opportunities to address these fundamental challenges and to develop technologies for muscle stem cell therapeutics to treat muscle disease. The proposed studies will enhance our understanding of fundamental mechanisms controlling muscle stem cell specification and differentiation in embryonic and regenerating muscles and have direct application to the development of stem cell technologies to regenerate damaged, aging, and diseased skeletal muscles. )